The present invention generally relates to MYB-like transcription factors that regulate the production of anthocyanin pigment. More specifically, the invention relates to PAP1 and PAP2 genes that can be used to increase pigmentation specifically in the petals, to identify plants under stress, and to upregulate the phenylpropanoid pathway.
Plants produce an enormous array of natural products. Ethnobotany and limited screens of medicinal plants indicate that among this huge repertoire of chemical diversity are many potentially useful bioactive structural principles for developing novel drugs, flavors, fragrances and other specialty chemicals. Unfortunately, these complex natural products usually occur at very low abundance and with a restricted tissue distribution. Moreover, almost all of this phytochemical biodiversity resides in exotic, uncultivated species. While important plant-based drugs such as Taxol(copyright), vinblastine, and vincristine have been discovered, the development of rational approaches for the generation of useful amounts of complex natural products for industrial evaluation remains an unsolved problem.
In particular, an intense 30-year effort on cell and tissue culture of medicinal plants has failed to generate useful levels of complex products either for the commercial production of established drugs in vitro, or for high throughput, multiplex screening of phytochemical biodiversity. This likely reflects the stringent spatial and temporal transcriptional controls governing the biosynthesis of specific natural products during plant development. Transgenic manipulation to override these genetic controls may thus provide the key to enhancing natural product biosynthesis for industrial evaluation and exploitation.
Activation tagging with the enhancer from the cauliflower mosaic virus (CaMV) 35S transcript promoter (35Se) is an emerging technology in plant functional genomics (Weigel, et al., Plant Physiol., 122:1003, 2000). This approach uses Agrobacterium tumefaciens-mediated transformation to create transgenic plants in which the transferred-DNA (T-DNA) containing the 35Se at its right border is spliced into the plant genome at random sites. In each independent transgenic line, the 35Se strongly activates the plant gene to which, by chance, it lies adjacent. Activation of a gene in this fashion often leads to observable effects on the modified plant, providing important clues about the function of the activated gene. Screening large collections of independent activation tagged lines thus represents a powerful way of surveying the genome and isolating genes affecting traits of interest.
A universal and well-studied flavonoid natural product is the floral pigment anthocyanin. The anthocyanin biosynthetic pathway is well characterized and many key regulators of the pathway have also been discovered. Genetic screens in Arabidopsis for loss of anthocyanin pigments were performed by screening for loss of pigmentation in the seed coat (testa). Many transparent testa loci correspond to anthocyanin biosynthetic genes. Transparent Testa Glaborous1 is a regulatory gene that encodes a WD40 repeat protein and whose mutant phenotype has pleiotropic defects including loss of anthocyanins and trichomes, and increased root hairs.
The WD40 repeat protein An11 in petunia also controls anthocyanin production. Anthocyanin production in many species is controlled by MYB and bHLH transcription factors. The maize MYB proteins C1 and pl interact with the bHLH proteins of the R/B family to regulate transcription of biosynthetic genes beginning with chalcone synthase (CHS). In petunia, the bHLH locus, An1 and Jaf13, and the MYB An2 locus, direct pigmentation. In Arabidopsis many bHLH and MYB genes exist but few have known functions. The MYB genes GL1 and CPC are involved in trichome and root hair initiation respectively. An Arabidopsis MYB gene involved in anthocyanin production heretofore had not been identified.
Embodiments of the invention provide an isolated PAP1 or PAP2 gene, or an active segment thereof. In some aspects, the invention provides a nucleic acid construct including a PAP1 or PAP2 gene, or an active segment thereof. In the construct the PAP1 or PAP2 gene can be operably linked to a promoter. The PAP1 gene can include the nucleotide sequence of SEQ ID NO:1; the PAP2 gene can include the nucleotide sequence of SEQ ID NO:3. The construct can encode a fusion protein including a PAP1 or PAP2 polypeptide, or an active segment thereof. The invention further provides a cell including the construct of this aspect of the invention. In another aspect, the invention provides a purified PAP1 or PAP2 polypeptide, or an active segment thereof. The polypeptide can include the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 and the polypeptide can be encoded by a nucleotide sequence including SEQ ID NO:1 or SEQ ID NO:3.
Another aspect of the invention is a transgenic plant including a nucleic acid construct having a PAP1 or PAP2 gene, or an active segment thereof. The gene can include the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; the plant can be Arabidopsis thaliana. The expression of the PAP1 or PAP2 gene can be tissue specific. In the transgenic plant, the tissue can be selected from the group consisting of, floral tissue, leaf, stem, root, cortex, meristem and cambium. PAP1, PAP2, or a segment thereof, can be overexpressed in floral organs, producing modified floral pigmentation, which can involve control of pigment production by action of a transposon.
In yet another aspect of the invention, there is provided a method of screening for gene expression including: providing a nucleic acid construct including a PAP1 or PAP2 gene or an active segment thereof, functionally fused to a gene of interest; transforming a plant cell with the nucleic acid construct to produce a transgenic plant or transgenic cell culture; and determining whether the gene of interest can be expressed based on the color of the transgenic plant or transgenic cell culture. The expression can be tissue-specific; the construct can include the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; the plant can be any plant amenable to transformation such as, for example, Arabidopsis thaliana. Another embodiment of this aspect of the invention is a method of identifying a gene in a biosynthetic pathway in a plant, including the steps of: transforming a population of plants or plant cells with a nucleic acid construct including an activation tag; screening the population to detect an individual having an increased accumulation of a product or an intermediate in the pathway; and identifying a gene of the biosynthetic pathway in the individual, based on proximity to the activation tag. The screening step can include chromatography to identify up-regulation of the product or intermediate in the pathway. A preferred mode of chromatography is HPLC. The product of the biosynthetic pathway can be a pigment, and the screening step can include screening the population to detect an individual having an increased production of a pigment, a pigment-related protein, or a pigment-related product, and the gene identified can be a pigment gene or a pigment-related gene. The pigment-related protein can be selected from the group consisting of: PAL1, PAL2, PAL3, CAD, 4CL, CHS, CHI, DFR, and flavone-3-hydrolase. The pigment-related product can be selected from the group consisting of: coumaric acid, coumarate conjugate, synapic acid, ferulic acid, salicylic acid, and lignin.
Aspects of the invention further provide a method of characterizing an intermediate in a biosynthetic pathway including: identifying a gene by an embodiment of the methods previously mentioned; transforming a plant or plant cell with the gene or a derivative thereof; and characterizing the intermediate by screening the transgenic plant or plant cell for up-regulated or down-regulated products. Also provided is a method of detecting a stress condition in a plant, including: providing a population of plants overexpressing a PAP1 or PAP2 gene or an active segment thereof; monitoring one or more plants of the population for color change associated with anthocyanin pigmentation; and detecting a stress condition in a plant, based on the color change. The stress condition can be associated with a stress selected from the group consisting of: light stress, water stress, pH stress, salt stress, temperature stress, heavy metal stress, pathogen attack or infection, wounding, nutrient deficiency, herbivory, and abnormal hormone levels.
Still other aspects of the invention provide a method of detecting promoter activity within a plant cell, including: providing a nucleic acid construct including a PAP1 or PAP2 gene, or an active segment thereof, operably linked to and promotable by a nucleic acid sequence including a promoter region or an active segment thereof; causing the construct to be expressed in the plant cell; and detecting a color or color change associated with anthocyanin pigmentation, wherein the color or color change arises from activity of the promoter region or active segment thereof within the plant cell. The plant cell can correspond to a tissue of a whole plant, and expression of the promoter can be specific to the tissue. Likewise, the plant cell can correspond to a developmental stage or structure of a whole plant, and expression of the promoter can be specific to the developmental stage or structure. Also provided is a method of screening for plant cell transformation, including the steps of: providing a nucleic acid construct including the gene of interest linked to a PAP1 or PAP2 gene; transforming a plurality of plant cells with the expression vector; and determining which of the plurality of plant cells were successfully transformed, by reference to altered color of the plant cells. The transformed plant cells can be used to produce transgenic plants, and the altered color expression can be tissue-specific in the transgenic plants.
Further aspects of the invention include is a biological preparation for the up-regulation of anthocyanin production including one or more peptides selected from the group consisting of PAP1, PAP2, and a mixture thereof, in a biologically acceptable, non-toxic vehicle. Also provided is a method for the enhanced accumulation of a phenylpropanoid product in a plant including: constructing a transgenic plant overexpressing PAP1 or PAP2, or an active segment thereof, whereby production of a plurality of phenylpropanoid products and intermediates in the plant can be elevated. The method can further include the step of causing expression within the transgenic plant of a gene involved in a step in production of a selected phenylpropanoid product, whereby elevated production of the plurality of phenylpropanoid products and intermediates favors enhanced accumulation of the selected product. The transgenic plant can be of a taxon that is characteristic for production of a selected phenylpropanoid product, and accumulation of the selected product can be enhanced by elevated production of the plurality of phenylpropanoid products and intermediates in the plant. For example, the plant can be Lycopersicon esculentum, and the selected product can be a lycopene; the plant can be Sesamum indicum, and the selected product can be sesamin and/or sesamolin; the plant can be any nitrogen fixing species, and wherein the selected product can be acetosyringone. In yet another aspect, the invention provides a system for expression of a gene of interest, the system including a PAP1 or PAP2 gene, or an active segment thereof, and a promoter including an anthocyanin myb binding site, wherein the promoter is operably linked to the gene of interest, wherein the product of the PAP1 or PAP2 gene or segment thereof binds to the myb binding site and causes expression of the gene of interest.